1. Field of the Invention
This invention relates to a DNA having genetic information of a human plasma-type glutathione peroxidase, a vector containing said DNA, a transformant holding said vector, a polypeptide produced by the expression of said DNA genetic information by the transformant.
2. Description of the Background
Glutathione peroxidase is an enzyme having glutathione as a substrate and catalyzing a reaction in which two moles of glutathione and one mole of hydrogen peroxide are converted into two moles of glutathione oxide and two moles of water. The enzyme is known to be present in tissues and organs of mammals, e.g. in liver, kidney, heart, lung, blood cells, plasma, and the like [Flohe et al., FEBS Lett., 32, 132-134 (9173)], and to play an important role in the disposition of in-vivo peroxides by catalyzing 2-electron reduction of lipid peroxides produced by glutathione. Thus, glutathione peroxidase acts to extinguish in-vivo hydrogen peroxide and lipid peroxides. In particular, the activity of glutathione peroxidase to extinguish the lipid peroxide is of interest in relation to various diseases caused by lipid peroxides, which are known to impart a strong action to damage cell membranes. Burn (scald), for example, increases lipid peroxides in serum and causes damage to various organs, resulting in increase in such values as GOT, LDH, alkali phosphatase, and the like. Increase in the amount of lipid peroxides in serum which is also observed in patients suffering from diabetes mellitus causes damage to blood vessels, which are considered to be a cause of arteriosclerosis and cerebral apoplexy. Moreover, as a direct action, lipid peroxides denature LDH (low density lipoprotein) in serum. Intake of LDH thus produced by macropharge causes foam cells to be produced which are considered to be a cause of arterial sclerosis.
Based on the above observations, glutathione peroxidase, which exhibits the lipid peroxide extinction activity, is considered to be useful for the treatment or the cure or the prevention of these diseases.
Glutathione peroxidase is a protein containing selenium, which exists in the active site in the form of selenocystein (Sec). The opal codon TGA, which normally is a termination codon in a DNA sequence, codes for Sec in a cloned glutathione peroxidase gene derived from mouse [EMBO Journal, vol 5, No. 6, 1221-1227 (1986)].
Human glutathione peroxidases are isolated from blood cells [J. Biol. Chem., 250, 5144-5149 (1975)], placenta [Bioch. J., 177, 471-476 (1979)], or liver [Chem. Pharm. Bull., 131, (1), 179-185 (1983)]0 as human blood cell-type glutathione peroxidase (hereinafter abbreviated as h-e.GSHPx). They are known to immunologically cross-over each other and to be composed of sub-units each having a molecular weight of about 20,600. The h-e.GSHPx was cloned from the cDNA library which was prepared from mRNA of kidney cells and liver cells, based on which the base sequence of their gene DNA have been determined [Nuc. Acids Res., 15, 10051 (1987); Nuc. Acids., 17, 7178 (1987)]. On the other hand, glutathione peroxidase was also confirmed to secrete in human plasma fractions. Sub-unit of this human plasma-type glutathione peroxidase (hereinafter abbreviated as h-p.GSHPx) were reported to exist as a homotetramer of glycoprotein having a molecular weight of 21,500-23,000 [Archivs of Biochemistry and Biophysics, 256, No. 2, 677-686 (1987); J. Biol. Chem., 1,262, No. 36, 17398-17403 (1987)]. The h-e.GSHPx derived from blood cells and h-p.GSHPx derived from plasma do not immunologically crossover each other, and thus are considered to be different enzymes from the aspect of protein chemistry. Even though at least two types of glutathione peroxidases, h-e.GSHPx and h-p.GSHPx, have so far been confirmed to exist, the latter has never been cloned.
In order to utilize h-p.GSHPx as a medicine for curing diseases caused by activated oxygen or hydrogen peroxide a large amount of purified h-p.GSHPx sample is required. At the present, however, the production of a large amount of h-p.GSHPx in a purified form is difficult and undesirable from the aspect of the safety.
The present inventors have isolated h-p.GSHPx from human plasma fractions according to a known method [Archivs of Biochemistry and Biophysics, 256, No. 2, 677-686 (1987)], and digested this purified enzyme sample with trypsin to obtain four peptide fragments. Amino acid sequences of these peptide fragments determined according to the Edman degradation method were:
-? ? ? -Gly-Leu-Thr-Gly-Gln-Tyr-Ile-Glu-Leu-Asn-Ala-Leu-Gln-; PA0 -Ala-Leu-Val-Ile-Leu-Gly-Phe-Pro-Cys-Asn-Gln-Phe-Gly-? ? ? -Gln-Glu-Pro-Asp-Glu-Asn-Ser-Glu-Ile-Leu-Pro-; PA0 -Thr-Phe-Leu-Asp-Asn-Ser-Phe-Pro-; and PA0 -Trp-Asn-Phe-Glu-? ? ? -Phe-Leu-Val-Gly-Pro-Asp-Gly-Ile-Pro-? ? ? -Met-Arg-,
wherein ? ? ? denotes a site where the amino acid could not be identified.
Triplet codons for a 13 amino acid sequence which is a portion of the polypeptide amino acid sequences thus determined were estimated, from which a probe mixture of 39 anti-codon bases was prepared. This DNA probe was used to screen a cDNA library of .lambda.-gt11 phage vector which was prepared from human placenta mRNA, thus identifying a gene consisting of 1,603 bases and encoding 225 amino acid residues. Analysis of the DNA sequence and the amino acid sequence proved that the selenocystein which is a site characteristic to glutathione peroxidase was encoded by an opal codon TGA, and the amino acid sequence in the neighborhood of the active center was -Ala-Ser-Tyr-***-Gly-Leu-Thr-, wherein *** denotes a selenocystein residue. The amino acid sequence had a homology of only about 30% with that of h-e.GSHPx, and the homology of the base sequence with that of h-e.GSHPx was only about 25%. Furthermore, the h-p.GSHPx was found to have in its N-terminal side, an amino acid sequence of the formula -Ile-Ser-Gly-Thr-Ile-, and in its C-terminal side, an amino acid sequence of the formula -Leu-Gly-Thr-Ser-Asp-. This gene DNA was recombined into an expression vector and transformed, for example, in monkey kidney cells (COS) to express a homotetramer which comprises a sub-unit having a molecular weight of 23,000.+-.2,000 and which could be discharged outside the cells by the action of the signal peptide which is characteristic to the amino acid sequence on the N-terminal side. The present inventors have thus prepared the h-p.GSHPx as well as its gene DNA, and have established a process for its preparation.